This is a competitive renewal proposal for R01 AG13038, currently in its 8' consecutive year of funding. The focus of this award has been to study the effects of aging and lifestyle interventions on large artery function and structure. In the present plan we propose to continue our productive work on this theme by testing the following tightly focused set of working hypotheses: 1) regular moderate-intensity aerobic exercise (daily brisk walking) increases peripheral conduit artery flow-mediated dilation (FMD), a measure of endothelium-dependent vasodilatory capacity and overall arterial vascular health, in previously sedentary middle-aged and older adults; 2) an increase in nitric oxide (NO) bioavailability is the key mechanism by which regular aerobic exercise improves FMD; 3) an increase in the bioavailability of the critical co-factor for NO synthesis, tetrahydrobiopterin (BH4), is one mechanism by which regular aerobic exercise increases NO bioavailability and FMD; 4) a reduction in vascular oxidative stress, related in part to an increase in extracellular superoxide dismutase (ecSOD), is an important mechanism by which regular aerobic exercise increases BH4 and NO bioavailability and FMD; 5) changes in the expression of proteins encoded by specific genes in arterial endothelial cells (i.e., increases in enzymatic antioxidant, eNOS, and phosphorylated eNOS protein expressions, and reductions in oxidant enzyme, endothelin-1, and angiotensin II receptor protein expressions) are among the key molecular mechanisms associated with the favorable effects of regular aerobic exercise on oxidative stress, BH4 and NO bioavailability, and FMD. To test these hypotheses we will conduct 2 complementary randomized aerobic exercise intervention trials in sedentary healthy middle-aged and older (age 55-75 years) men and women. The mechanistic roles played by changes in vascular oxidative stress and BH4 and NO bioavailability in mediating improvements in FMD will be determined in experimental sessions conducted before and after a 12-week exercise (or non-exercise attention control) condition. Insight into the molecular mechanisms involved will be obtained using a novel translational physiology research technique by which changes in arterial endothelial cell protein expression of genes involved in the regulation of these cellular and systemic adaptations to habitual exercise will be determined via quantitative immunofluorescence. The expected results will provide new, clinically important insight into the efficacy of moderate aerobic exercise for restoring arterial endothelial function in middle-aged and older sedentary adults, and the underlying mechanisms. In particular, the proposed research will provide the first information on 2 highly novel mechanisms by which regular exercise may augment NO bioavailability: 1) by increasing BH4 bioavailability; and 2) by producing changes in the expression of key arterial endothelial cell proteins involved in determining endothelial function. :_OVIDED. University of Colorado-Boulder, Boulder, Colorado